Chemical cleaning of biological material

ABSTRACT

The invention is directed to collagenous tissues which have been treated to remove non-collagenous components such as cells, cellular debris, and other extracellular matrix components, such as proteoglycans and glycosaminoglycans, normally found in native tissues. Treatment of the tissue with alkali, chelating agents, acids and salts removes non-collagenous components from the collagenous tissue matrix while controlling the amount of swelling and dissolution so that the resultant collagen matrix retains its structural organization, integrity and bioremodelable properties. The process circumvents the need to use detergents and enzymes which detrimentally affect the cell compatibility, strength and bioremodelability of the collagen matrix. The collagenous tissue matrix is used for implantation, repair, or use in a mammalian host.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 08/853,372, filedMay 8, 1997, which issued as U.S. Pat. No. 5,993,844.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is in the field of tissue engineering. The invention isdirected to collagenous tissues which have been treated to removenon-collagenous components such as cells, cellular debris, and otherextracellular matrix components, such as proteoglycans andglycosaminoglycans, normally found in native tissues. Treatment of thetissue with alkali, chelating agents, acids and salts removesnon-collagenous components from the collagenous tissue matrix whilecontrolling the amount of swelling and dissolution so that the resultantcollagen matrix retains its structural organization, integrity andbioremodelable properties. The process circumvents the need to usedetergents and enzymes which detrimentally affect the cellcompatibility, strength and bioremodelability of the collagen matrix.The collagenous tissue matrix is used for implantation, repair, or usein a mammalian host.

2. Brief Description of the Background of the Invention

The field of tissue engineering combines the methods of the engineeringwith the principles of life sciences to understand the structural andfunctional relationships in normal and pathological mammalian tissues.The goal of tissue engineering is the development and ultimateapplication of biological substitutes to restore, maintain or improvetissue functions. [Skalak, R. and Fox, C. F., “Tissue Engineering”, AlanR. Liss Inc. N.Y. (1988)].

Collagen is the principal structural protein in the body and constitutesapproximately one-third of the total body protein. It comprises most ofthe organic matter of the skin, tendons, bones and teeth and occurs asfibrous inclusions in most other body structures. Some of the propertiesof collagen are its high tensile strength; its ion exchanging ability,due in part to the binding of electrolytes, metabolites and drugs; itslow antigenicity, due to masking of potential antigenic determinants bythe helical structure, and its low extensibility, semipermeability, andsolubility. Furthermore collagen is a natural substance for celladhesion. These properties and others make collagen a suitable materialfor tissue engineering and manufacture of implantable biologicalsubstitutes and bioremodelable prostheses.

As collagen is one major component of these biological substitutes, amethod for obtaining sufficient quantities of collagen that isconsistent in quality is needed. A need currently exists for an improvedmethod for the removal of non-collagenous components such as cells,cellular debris, and other extracellular matrix components, such asproteoglycans and glycosaminoglycans, normally found in native tissuesto yield a substantially pure native collagen matrix. Some of thesenon-collagenous structures that are present in native tissues arebelieved to be antigenic and will elicit a chronic inflammatory responsewhen implanted in a host. However, in the art there are a variety ofmethods for the cleaning of such collagenous tissue which have resultedin collagenous compositions with different characteristics. The methodused should be one that maintains the biological and physical propertiesof collagen and collagenous tissues suitable for use in tissueengineering.

In the art of treating a collagenous tissue to yield essentially acollagenous matrix, detergents and surfactants have customarily beenused in the extraction of cells and lipids from the tissue. Detergentssuch as sodium dodecyl sulfate (SDS) are amphipathic molecules whereinthe hydrophobic region binds to protein and are believed to increase thenegative charge of the protein. When implanted, the increase in chargeresults in both the swelling of the tissue due to increased waterbinding by the hydrophilic region of the molecule, and decreased thermalstability in collagen by disrupting hydrogen bonding. Swelling bothopens the structure of the collagen molecule making it susceptible tocellular enzymes such as collagenase and destabilizes the collagenmatrix to result in a weakened construct (Courtman, et al. Journal ofBiomedical Materials Research 1994; 28:655-666.) It is further believedthat SDS residues remain bound to the collagen and prevent cells frommigrating into the implant. (Wilson, G J et al Ann Thorac Surg 1995;60:S353-8. Bodnar E, et al. “Damage of aortic valve tissue caused by thesurfactant sodium dodecyl sulfate.” Thorac Cardiovasc Surg 1986;34:82-85.) Because detergents used in a chemical cleaning method canundesirably bind to and alter the bioremodeling capabilities of collagenin the treated tissue, the inventors have developed a method thateliminates the need for detergents.

Chemical cleaning of tissue with enzymes such as trypsin, pepsin andcollagenase is known in the art but their use will result in chemicalmodification of the native collagen molecules and will adversely affectthe structural integrity of the construct. Enzyme treatment ofcollagenous tissue is known in the art for removal and/or modificationof extracellular matrix associated proteins. Proteases such as pepsin,trypsin, dispase, or thermolysin are used in the removal of collagentelopeptides to yield atelopeptide collagen. Collagen telopeptides arethe non-triple helical portion of the collagen molecule and have beenthought by some researchers to be weakly antigenic while by others theyare thought to be responsible for the strong mechanical properties ofcollagen. Limited digestion of collagenous tissue will removetelopeptides without dissociation of the collagen matrix of the tissue,while prolonged digestion will dissociate the collagen fibrils intoatelopeptide collagen monomers. It is also known in the art to modifyand remove nucleic acids from the matrix using enzymes that digestendogenous RNA and DNA through use of RNAse and DNAse, respectively. Astreatment with enzymes can affect the structural integrity of thecollagen, the present method of the invention circumvents their use.

Methods for obtaining collagenous tissue and tissue structures fromexplanted mammalian tissue, and processes for constructing prosthesesfrom the tissue, have been widely investigated for surgical repair orfor tissue and organ replacement The tissue is typically treated toremove potentially cytotoxic cellular and noncollagenous components toleave a natural tissue matrix. Further processing, such as crosslinking,disinfecting or forming into shapes have also been investigated.Previous methods for treating collagenous tissue to remove tissuecomponents from the organized tissue matrix have employed detergents,enzymes or promote uncontrolled swelling of the matrix. WO 95/28183 toJaffe, et al. discloses methods to decrease or prevent bioprostheticheart valve mineralization postimplantation. The disclosed methodsprovide biological material made acellular by controlled autolysis.Autolysis is controllably performed using at least one buffer solutionat a preselected pH to allow autolytic enzymes present in the tissue todegrade cellular structural components. U.S. Pat. No. 5,007,934 to Stoneand, similarly, U.S. Pat. No. 5,263,984 to Li, et al. both disclose amultiple step method for chemical cleaning of ligamentous tissue. Themethod utilizes a detergent to remove lipids associated with cellmembranes or collagenous tissue. U.S. Pat. No. 5,523,291 to Janzen, etal. discloses an comminuted injectable implant composition for softtissue augmentation derived from ligamentum nuchae. The ligament istreated with a series soaks in a strongly alkaline solution of sodiumhydroxide followed by hydrochloric acid solution and then sodiumbicarbonate. U.S. Pat. No. 5,028,695 to Eckmayer, et al. discloses aprocess for the manufacture of collagen membranes in which collagenoustissue is repeatedly treated with a strong alkali and subsequently witha strong acid for a number of times then further treated with inorganicsaline treatment to shrink the membranes and then with solvent to drythem.

SUMMARY OF THE INVENTION

Bioremodelable collagenous tissue matrices and methods for chemicalcleaning of native tissue to produce such tissue matrices are disclosed.

The present invention overcomes the difficulties in obtainingbioremodelable tissue matrices that are substantially collagen. Theinvention provides tissue matrices that can be used as a prostheticdevice or material for use in the repair, augmentation, or replacementof damaged and diseased tissues and organs.

The chemical cleaning method of this invention renders biologicalmaterial, such as native tissues and tissue structures, substantiallyacellular and substantially free of non-collagenous components whilemaintaining the structural integrity of the collagenous tissue matrix.As detergents are not used in the chemical cleaning process, detergentresidues that would normally remain bound to the tissue matrix are notpresent. As enzymes are not used, the collagen telopeptides are retainedon the collagen molecules. The method comprises contacting a normallycellular native tissue with a chelating agent at a basic pH, contactingthe tissue with salt solution at an acidic pH, contacting the tissuewith a salt solution at a physiologic pH, and, then finally rinsing theresultant chemically cleaned tissue matrix.

This invention is directed to a chemically cleaned tissue matrix derivedfrom native, normally cellular tissues. The cleaned tissue matrix isessentially collagen rendered substantially free of glycoproteins,glycosaminoglycans, proteoglycans, lipids, non-collagenous proteins andnucleic acids such as DNA and RNA. Importantly, the bioremodelability ofthe tissue matrix is preserved as it is free of bound detergent residuesthat would adversely affect the bioremodelability of the collagen.Further the collagen is telopeptide collagen as the telopeptide regionsof the collagen molecules remain intact as it has not undergonetreatment or modification with enzymes during the cleaning process.

The collagenous material generally maintains the overall shape of thetissue it is derived from but it may be layered and bonded together toform multilayer sheets, tubes, or complex shaped prostheses. The bondedcollagen layers of the invention are structurally stable, pliable,semi-permeable, and suturable. When the matrix material is implantedinto a mammalian host, it undergoes biodegradation accompanied byadequate living cell replacement, or neo-tissue formation, such that theoriginal implanted material is ultimately remodeled and replaced by hostderived tissue and cells.

It is, therefore, an object of this invention to provide a method forcleaning native tissue resulting in a tissue matrix that does notexhibit many of the shortcomings associated with many of the methodsdeveloped previously. The method effectively removes non-collagenouscomponents of native tissue without the use of detergents or enzymes toyield a tissue matrix comprised substantially of collagen.

Another object is the provision of a bioremodelable tissue matrixmaterial that will allow for and facilitate tissue ingrowth and/or organregeneration at the site of implantation. Prostheses prepared from thismaterial, when engrafted to a recipient host or patient, concomitantlyundergoes controlled bioremodeling and adequate living cell replacementsuch that the original implanted prosthesis is remodeled by thepatient's living cells to form a regenerated organ or tissue.

Still another object of this invention is to provide a method for use ofa novel multi-purpose bioremodelable matrix material in autografting,allografting, and heterografting indications.

Still a further object is to provide a novel tissue matrix material thatcan be implanted using conventional surgical techniques.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for processing nativecollagenous tissues for transplantation. The processing method isdesigned to generate an implantable, graftable collagenous biologicaltissue material, an extracellular matrix comprising collagen, thatserves as a scaffold that can be bioremodeled by a host in vivo or byliving cells in culture in vitro.

This invention is further directed to a tissue engineered prosthesesformed from processed native collagenous tissue, which, when implantedinto a mammalian host, can serve as a functioning repair, augmentation,or replacement body part, or tissue structure, and will undergocontrolled biodegradation occurring concomitantly with remodeling by thehost's cells. The tissue matrix can be used as a prosthetic material forautografting, allografting, and heterografting indications. Theprosthesis of this invention, in its various embodiments, thus has dualproperties: First, it functions as a substitute body part, and second,while still functioning as a substitute body part, it functions as aremodeling template for the ingrowth of host cells. Although theprostheses will be illustrated through construction of various devicesand constructs, the invention is not so limited. It will be appreciatedthat the device design in its material, shape and thickness is to beselected depending on the ultimate indication for the construct.

The chemical cleaning method of this invention renders biologicalmaterial, such as native tissues and tissue structures, substantiallyacellular and substantially free of non-collagenous components whilemaintaining the structural integrity of the collagenous tissue matrix.Elastin is sometimes present in native tissue in small amounts and isnot removed by the chemical cleaning method. The presence of elastin maybe desirable for certain applications. As used herein, the term,“substantially acellular” means having at least 95% fewer native cellsand cell structures than the natural state of the biological material.“Cells and cellular structures” refer to cells, living or not living,cell remnants, cell membranes and membrane structures. By use of theterm, “substantially free of non-collagenous components”, Applicantsmean that glycoproteins, glycosaminoglycans, proteoglycans, lipids,non-collagenous proteins and nucleic acids such as DNA and RNA compriseless than 5% of the resultant tissue matrix. As detergents are not usedin the chemical cleaning process, detergent residues that would normallyremain bound to the tissue matrix are not present. As enzymes are notused, the collagen telopeptides are retained on the collagen molecules.Further, the chemical cleaning method renders the biological materialboth sterile and endotoxin free when processed using sterile equipment,solutions and aseptic technique.

The term, “structural integrity”, refers to the capacity of thechemically cleaned collagenous tissue matrix to withstand forces such astension, compression, and support The structural integrity of thebiological material is preserved as swelling is minimized in thechemical treatment steps even though some swelling will occur duringtreatment Uncontrolled or excessive swelling both opens the structure ofthe collagen molecule making it susceptible to cellular enzymes such ascollagenase and destabilizes the collagen to result in a weakenedconstruct. As swelling affects the intramolecular structure of thecollagen molecule, it affects the overall structure of the material on aintermolecular level by disrupting the native crosslinks betweencollagen molecules. Together, the structure of the collagen molecule andthe crosslinks between collagen molecules lend structural integrity tothe material.

Tissue matrix material that maintains much of its native structuralintegrity is useful, for instance, when used as a prosthetic device oras material to construct mulitilayered or complex devices. The integrityof the material is important if it is to perform a load bearing functionsuch as a body wall support, a vascular device, or an orthopedic device.Related to structural integrity is the term “suturable” which means thatthe mechanical properties of the material includes suture retentionwhich permits needles and suture materials to pass through theprosthesis material at the time of suturing of the prosthesis tosections of native tissue, a process known as anastomosis. Duringsuturing, such prostheses must not tear as a result of the tensileforces applied to them by the suture, nor should they tear when thesuture is knotted. Suturability of the prosthetic material, i.e., theability of prostheses to resist tearing while being sutured, is relatedto the-intrinsic mechanical strength of the prosthesis material, thethickness of the graft, the tension applied to the suture, and the rateat which the knot is pulled closed.

Biological material as defined in the invention includes but is notlimited to harvested mammalian tissues, and structures thereof, derivedfrom human, bovine, porcine, canine, ovine, caprine, and equineorganisms. Tissue structures such as dermis, artery, vein, pericardium,heart valve, dura mater, ligament, intestine and fascia are allpreferred tissue structures that are able to be cleaned by the methodsof this invention to yield a tissue matrix that is substantiallyacellular and substantially free of non-collagenous components.

A preferred source of mammalian tissue is the tunica submucosa fromsmall intestine, most preferably from porcine small intestine. In nativesmall intestine, the tunica submucosa is the connective tissue layer ofthe organ and comprises both lymphatic and blood vessel cells. Methodsfor obtaining tunica submucosa are disclosed in WO 96/31157 and isincorporated herein. To obtain porcine tunica submucosa, also termed“submucosa”, the small intestine of a pig is harvested and mechanicallystripped, preferably by use of a gut cleaning machine (Bitterling,Nottingham, UK). The gut cleaning machine forcibly removes the fat,muscle and mucosal layers from the tunica submucosa using a combinationof mechanical action and washing with water. The mechanical action canbe described as a series of rollers that compress and strip away thesuccessive layers from the tunica submucosa when the intact intestine isrun between them. As the tunica submucosa of the small intestine iscomparatively harder and stiffer than the surrounding tissue, the softercomponents from the submucosa are removed from the tunica submucosa. Theresult of the machine cleaning is such that the mesenteric tissues, thetunica serosa and the tunica muscularis from the ablumen of the tunicasubmucosa and as well as the layers of the tunica mucosa from the lumenof the tunica submucosa are removed from the tunica submucosa so thatthe tunica submucosa layer of the intestine solely remains. Thechemically cleaned tissue matrix of the tunica submucosa is also termed“intestinal collagen layer” or “ICL”. It is noted that in some animalsources, such as carnivores and omnivores, the small intestine includesa stratum compactum which is also removed by this mechanical cleaningstep.

Other methods of mechanically stripping layers of the small intestineare known in the art as described in U.S. Pat. No. 4,902,508 to Badylak,incorporated herein by reference. The method disclosed by this patentincludes mild abrasion of the intestinal tissue to remove the abluminallayers, including the tunica serosa and the tunica muscularis, and theinner layers consisting of at least the luminal portion of the tunicamucosa. The layers that remain are the tunica submucosa with theattached basilar layer consisting of lamina muscularis mucosa and, ifinitially present in the harvested mammalian tissue, stratum compactum.Intestinal material obtained by either method can be implanted or firstformed into body wall or vascular device by a number of methodsincluding suturing, stapling. adhesive compositions, chemical bondingand thermal bonding.

Terms pertaining to certain operating parameters are defined for theentire specification and the examples for amounts, times andtemperatures that can be varied without departing from the spirit andscope of the invention. As used herein, an “effective amount” refers tothe volume and concentration of composition required to obtain theeffect desired. A preferred effective amount for the chemical cleaningof tissue is a ratio of 100:1 v/v of solution to tissue but volumes moreor less can be determined by the skilled artisan when considering theshape, bulk, thickness, density, and cellularity of the tissue to becleaned. The time required for the chemical steps to be effective can beappreciated by those of skill in the art when considering thecellularity, matrix density, and thickness of the material to becleaned. Larger, thicker, or denser materials will take longer for thesolutions to penetrate and equilibrate in tissue. The temperatures forthe environment and the solutions used in the present invention ispreferably at ambient room temperature, about 25° C., but can beanywhere in the range of above the freezing temperatures of thesolutions used to less than the denaturation temperature of the tissuematerial being treated. Temperatures between about 4° C. to about 45° C.are sufficient for the cleaning treatment to be effective. Agitation ismeant to be mechanical shaking or mixing and is used to improve thepenetration of the chemical compositions into the tissue and to reducethe time needed for chemical treatment to be effective. The term“buffered solution” refers to an aqueous solution containing at leastone agent which preserves the hydrogen ion concentration or pH of thesolution.

In the preferred method, harvested tissue may need to be cleanedmanually, as by gross dissection, and/or mechanically cleaned of excesstissues such as fat and vasculature. Manual cleaning may be necessaryfor some tissues for handling manageability during processing or formost effective chemical treatment.

The tissue is first treated by contacting the tissue with an effectiveamount of chelating agent, preferably physiologically alkaline tocontrollably limit swelling of the tissue matrix. Chelating agentsenhance removal of cells, cell debris and basement membrane structuresfrom the matrix by reducing divalent cation concentration. Alkalinetreatment dissociates glycoproteins and glycosaminoglycans from thecollagenous tissue and saponifies lipids. Chelating agents known in theart which may be used include, but are not limited to,ethylenediaminetetraacetic acid (EDTA) andethylenebis(oxyethylenitrilo)tetraacetic acid (EGTA). EDTA is apreferred chelating agent and may be made more alkaline by the additionof sodium hydroxide (NaOH), calcium hydroxide Ca(OH)₂, sodium carbonateor sodium peroxide. EDTA or EGTA concentration is preferably betweenabout 1 to about 200 mM; more preferably between about 50 to about 150mM; most preferably around about 100 mM. NaOH concentration ispreferably between about 0.001 to about 1 M; more preferably betweenabout 0.001 to about 0.10 M; most preferably about 0.01 M. Other alkaneor basic agents can be determined by one of skill in the art to bringthe pH of the chelating solution within the effective basic pH range.The final pH of the basic chelating solution should be preferablybetween about 8 and about 12, but more preferably between about 11.1 toabout 11.8. In the most preferred embodiment, the tissue is contactedwith a solution of 100 mM EDTA/10 mM NaOH in water. The tissue iscontacted preferably by immersion in the alkaline chelating agent whilemore effective treatment is obtained by agitation of the tissue and thesolution together for a time for the treatment step to be effective.

The tissue is then contacted with an effective amount of acidicsolution, preferably containing a salt. Acid treatment also plays a rolein the removal of glycoproteins and glycosaminoglycans as well as in theremoval of non-collagenous proteins and nucleic acids such as DNA andRNA. Salt treatment controls swelling of the collagenous tissue matrixduring acid treatment and is involved with removal of some glycoproteinsand proteoglycans from the collagenous matrix. Acid solutions known inthe art may be used and may include but are not limited to hydrochloricacid (HCl), acetic acid (CH₃COOH) and sulfuric acid (H₂SO₄). A preferredacid is hydrochloric acid (HCl) at a concentration preferably betweenabout 0.5 to about 2 M, more preferably between about 0.75 to about 1.25M; most preferably around 1 M. The final pH of the acid/salt solution ispreferably between about 0 to about 1, more preferably between about 0and 0.75, and most preferably between about 0.1 to about 0.5.Hydrochloric acid and other strong acids are most effective for breakingup nucleic acid molecules while weaker acids are less effective. Saltsthat may be used are preferably inorganic salts and include but are notlimited to chloride salts such as sodium chloride (NaCl), calciumchloride (CaCl₂), and potassium chloride (KCl) while other effectivesalts may be determined by one of skill in the art Preferably chloridesalts are used at a concentration preferably between about 0.1 to about2 M; more preferably between about 0.75 to about 1.25 M; most preferablyaround 1 M. A preferred chloride salt for use in the method is sodiumchloride (NaCl). In the most preferred embodiment, the tissue iscontacted with 1 M HCl/1 M NaCl in water. The tissue is contactedpreferably by immersion in the acid/salt solution while effectivetreatment is obtained by agitation of the tissue and the solutiontogether for a time for the treatment step to be effective.

The tissue is then contacted with an effective amount of salt solutionwhich is preferably buffered to about a physiological pH. The bufferedsalt solution neutralizes the material while reducing swelling. Saltsthat may be used are preferably inorganic salts and include but are notlimited to chloride salts such as sodium chloride (NaCl), calciumchloride (CaCl₂), and potassium chloride (KCl); and nitrogenous saltssuch as ammonium sulfate (NH₃SO₄) while other effective salts may bedetermined by one of skill in the art. Preferably chloride salts areused at a concentration preferably between about 0.1 to about 2 M; morepreferably between about 0.75 to about 1.25 M; most preferably about 1M. A preferred chloride salt for use in the method is sodium chloride(NaCl). Buffering agents are known in the art and include but are notlimited to phosphate and borate solutions while others can be determinedby the skilled artisan for use in the method. One preferred method tobuffer the salt solution is to add phosphate buffered saline (PBS)preferably wherein the phosphate is at a concentration from about 0.001to about 0.02 M and a salt concentration from about 0.07 to about 0.3 Mto the salt solution. A preferred pH for the solution is between about 5to about 9, more preferably between about 7 to about 8, most preferablybetween about 7.4 to about 7.6. In the most preferred embodiment, thetissue is contacted with 1 M sodium chloride (NaCl)/10 mM phosphatebuffered saline (PBS) at a pH of between about 7.0 to about 7.6. Thetissue is contacted preferably by immersion in the buffered saltsolution while effective treatment is obtained by agitation of thetissue and the solution together for a time for the treatment step to beeffective.

After chemical cleaning treatment, the tissue is then preferably rinsedfree of chemical cleaning agents by contacting it with an effectiveamount of rinse agent. Agents such as water, isotonic saline solutionsand physiological pH buffered solutions can be used and are contactedwith the tissue for a time sufficient to remove the cleaning agents. Apreferred rinse solution is physiological pH buffered saline such asphosphate buffered saline (PBS). Other means for rinsing the tissue ofchemical cleaning agents can be determined by one of skill in the art.The cleaning steps of contacting the tissue with an alkaline chelatingagent and contacting the tissue with a acid solution containing salt maybe performed in either order to achieve substantially the same cleaningeffect. The solutions may not be combined and performed as a singlestep, however.

A preferred composition of the invention is a chemically cleaned tissuematrix derived from native, normally cellular tissues. The cleanedtissue matrix is essentially acellular telopeptide collagen, about 93%by weight, with less than about 5% glycoproteins, glycosaminoglycans,proteoglycans, lipids, non-collagenous proteins and nucleic acids suchas DNA and RNA. Importantly, the bioremodelability of the tissue matrixis preserved as it is free of bound detergent residues that wouldadversely affect the bioremodelability of the collagen. Additionally,the collagen molecules have retained their telopeptide regions as thetissue has not undergone treatment with enzymes during the cleaningprocess.

Tissue matrices are derived from dermis, artery, vein, pericardium,heart valves, dura mater, ligaments, intestine and fascia A mostpreferred composition is a chemically cleaned intestinal collagen layerderived from the small intestine. Suitable sources for small intestineare mammalian organisms such as human, cow, pig, sheep, dog, goat orhorse while small intestine of pig is the preferred source. In onepreferred embodiment, the collagen layer comprises the tunica submucosaderived from porcine small intestine. In another embodiment, thecollagen layer comprises the tunica submucosa and the basilar layers ofthe small intestine. The basilar layers consist of lamina muscularismucosa and, if present in the native tissue, the stratum compactum.

The most preferred composition of the invention is the intestinalcollagen layer, cleaned by the chemical cleaning method of theinvention, which is essentially collagen, primarily Type I collagen,with less than about 5% glycoproteins, glycosaminoglycans,proteoglycans, lipids, non-collagenous proteins and nucleic acids suchas DNA and RNA. The collagen layer is free of bound detergent residuesthat would adversely affect the bioremodelability of the collagen. Thecollagen layer is substantially free of cells and cellular debris,including endogenous nucleic acids such as DNA and RNA and lipids.Further, the intestinal collagen layer is both sterile and endotoxinfree when processed using sterile equipment, solutions and aseptictechnique. Further, the intestinal collagen layer is both sterile andendotoxin free when processed using sterile equipment, solutions andaseptic technique.

Once the collagenous tissue matrix has been rendered substantiallyacellular and free of substantially noncollagenous extracellular matrixcomponents, prostheses for implantation or engraftment may bemanufactured therefrom. Collagen layers may be sutured or bondedtogether by use of any variety of techniques known in the art. Methodsfor bonding the layers may employ adhesives such as thrombin, fibrin orsynthetic materials such as cyanomethacrylates or chemical crosslinkingagents. Other methods may employ heat generated by laser, light, ormicrowaves. Convection ovens and heated liquid baths may also beemployed.

Thermal welding of the collagen layers is the preferred method forbonding together the collagen layers of the invention. Methods forthermal welding of collagen are described in WO 95/22301, WO 96/31157and U.S. Pat. No. 5,571,216, the teachings of which are incorporatedherein by reference. The ICL is first cut longitudinally and flattenedonto a solid, flat plate. One or more successive layers are thensuperimposed onto one another, preferably in alternating perpendicularorientation. A second solid flat plate is placed on top of the layersand the two plates are clamped tightly together. The complete apparatus,clamped plates and collagen layers, are then heated for a time and underconditions sufficient to effect the bonding of the collagen layerstogether. The amount of heat applied should be sufficiently high toallow the collagen to bond, but not so high as to cause the collagen toirreversibly denature. The time of the heating and bonding will dependupon the type of collagen material layer used, the moisture content andthickness of the material, and the applied heat. A typical range of heatis from about 50° C. to about 75° C., more typically 60° C. to 65° C.and most typically 62° C. A typical range of times will be from about 7minutes to about 24 hours, typically about one hour. The degree of heatand the amount of time that the heat is applied can be readilyascertained through routine experimentation by varying the heat and timeparameters. The bonding step may be accomplished in a conventional oven,although other apparatus or heat applications may be used including, butnot limited to, a water bath, laser energy, or electrical heatconduction. Immediately following the heating and bonding, the collagenlayers are cooled, in air or a water bath, at a range between roomtemperature at 20° C. and 1° C. Rapid cooling, termed quenching, isrequired to stop the heating action and to create an effective bondbetween the collagen layers. To accomplish this step, the collagenlayers may be cooled, typically in a water bath, with a temperaturepreferably between about 1° C. to about 10° C., most preferably about 4°C. Although cooling temperatures below 1° C. may be used, care will needto be taken not to freeze the collagen layers, which may causestructural damage. In addition, temperatures above 10° C. may be used inquenching, but if the temperature of the quench is too high, then therate of cooling may not be sufficient to fix the collagen layers to oneanother.

In the preferred embodiment, the collagenous material is crosslinked.Crosslinking imparts increased strength and structural integrity to theformed prosthetic construct while regulating the bioremodeling of thecollagen by cells when the construct is implanted into a patient.Collagen crosslinking agents include glutaraldehyde, formaldehyde,carbodiimides, hexamethylene diisocyanate, bisimidates, glyoxal, adipylchloride, dialdehyde starch, and certain polyepoxy compounds such asglycol diglycidyl ether, polyol polyglycidyl ether and dicarboxylic aciddiglycidylester. Dehydrothermal, UV irradiation and/or sugar-mediatedmethods may also be used. Collagen will also naturally crosslink withage standing at room temperature. However, crosslinking agents need notbe limited to these examples as other crosslinking agents and methodsknown to those skilled in the art may be used. Crosslinking agentsshould be selected so as to produce a biocompatible material capable ofbeing remodeled by host cells. A preferred crosslinking agent is1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Thecrosslinking solution containing EDC and water may also contain acetone.Crosslinking with EDC had been described in International PCTPublication Nos. WO 95/22301 and WO 96/31157.

In some embodiments, additional collagenous layers may be added toeither the outer or inner surfaces of the bonded collagen layers, eitherbefore or after crosslinking. In tubular constructs, as in a vascularconstruct, dense fibrillar collagen may be added to the luminal surfaceto create a smooth flow surface for its ultimate application asdescribed in International PCT Publication No. WO 95/22301, incorporatedherein by reference. This smooth collagenous layer also promotes hostcell attachment, as in the formation of neointima, which facilitatesingrowth and bioremodeling of the construct As described inInternational PCI Publication No. WO 95/22301, this smooth collagenouslayer may be made from acid-extracted fibrillar or non-fibrillarcollagen, which is predominantly type I collagen, but may also includeother types of collagen. The collagen used may be derived from anynumber of mammalian sources, typically bovine, porcine, or ovine skin ortendons. The collagen preferably has been processed by acid extractionto result in a fibril dispersion or gel of high purity. Collagen may beacid-extracted from the collagen source using a weak acid, such asacetic, citric, or formic acid. Once extracted into solution, thecollagen can be salt-precipitated using NaCl and recovered, usingstandard techniques such as centrifugation or filtration. Details ofacid extracted collagen from bovine tendon are described, for example,in U.S. Pat. No. 5,106,949, incorporated herein by reference.

Heparin can be applied to the prosthesis, by a variety of well-knowntechniques. For illustration, heparin can be applied to the prosthesisin the following three ways. First, benzalkonium heparin (BA-Hep)solution can be applied to the prosthesis by dipping the prosthesis inthe solution and then air-drying it. This procedure treats the collagenwith an ionically bound BA-Hep complex. Second, EDC can be used toactivate the heparin, then to covalently bond the heparin to thecollagen fiber. Third, EDC can be used to activate the collagen, thencovalently bond protamine to the collagen and then ionically bondheparin to the protamine. Many other coating, bonding, and attachmentprocedures are well known in the art which could also be used.

Treatment of the tissue matrix material with agents such as growthfactors or pharmaceuticals in addition to or in substitution for heparinmay be accomplished. The agents may include for example, growth factorsto promote vascularization and epithelialization, such as macrophagederived growth factor (MDGF), platelet derived growth factor (PDGF),vascular endothelial cell derived growth factor (VEGF); antibiotics tofight any potential infection from the surgery implant; or nerve growthfactors incorporated into the inner collagenous layer when theprosthesis is used as a conduit for nerve regeneration. In addition toor in substitution for drugs, matrix components such as proteoglycans orglycoproteins or glycosaminoglycans may be included within theconstruct.

The collagenous prosthesis thus formed can also be sterilized in adilute peracetic acid solution with a neutral pH. Methods forsterilizing collagen are described U.S. Pat. No. 5,460,962 and areincorporated by reference herein. In the preferred method, the collagenis disinfected with a dilute peracetic acid solution at a neutral pH.The peracetic acid concentration is preferably between about 0.01 and0.3% v/v in water at a neutralized pH between about pH 6 and pH 8.Alternatively, sterilization with gamma irradiation, at typically 2.5Mrad, or with gas plasma can also be used to sterilize the collagen.Other methods known in the art for sterilizing collagen may also beused.

The following examples are provided to better explain the practice ofthe present invention and should not be interpreted in any way to limitthe scope of the present invention. Those skilled in the art willrecognize that various modifications can be made to the methodsdescribed herein while not departing from the spirit and scope of thepresent invention.

EXAMPLES Example 1 Chemical Cleaning of Mechanically Stripped PorcineSmall Intestine

The small intestine of a pig was harvested and mechanically stripped,using a Bitterling gut cleaning machine (Nottingham, UK) which forciblyremoves the fat, muscle and mucosal layers from the tunica submucosausing a combination of mechanical action and washing using water. Themechanical action can be described as a series of rollers that compressand strip away the successive layers from the tunica submucosa when theintact intestine is run between them. The tunica submucosa of the smallintestine is comparatively harder and stiffer than the surroundingtissue, and the rollers squeeze the softer components from thesubmucosa. The result of the machine cleaning was such that thesubmucosal layer of the intestine solely remained. The remainder of theprocedure was performed under aseptic conditions and at roomtemperature. The chemical solutions were all used at room temperature.The intestine was then cut lengthwise down the lumen and then cut into15 cm sections. Material was weighed and placed into containers at aratio of about 100:1 v/v of solution to intestinal material.

A. To each container containing intestine was added approximately 1 Lsolution of 0.22 mm (micron) filter sterilized 100 mMethylenediaminetetraacetic tetrasodium salt (EDTA)/10 mM sodiumhydroxide (NaOH) solution. Containers were then placed on a shaker tablefor about 18 hours at about 200 rpm. After shaking, the EDTA/NaOHsolution was removed from each bottle.

B. To each container was then added approximately 1 L solution of 0.22mm filter sterilized 1 M hydrochloric acid (HCl)/1 M sodium chloride(NaCl) solution. Containers were then placed on a shaker table forbetween about 6 to 8 hours at about 200 rpm. After shaking, the HCl/NaClsolution was removed from each container.

C. To each container was then added approximately 1 L solution of 0.22mm filter sterilized 1 M sodium chloride (NaCl)/10 mM phosphate bufferedsaline (PBS). Containers were then placed on a shaker table forapproximately 18 hours at 200 rpm. After shaking, the NaCl/PBS solutionwas removed from each container.

D. To each container was then added approximately 1 L solution of 0.22mm filter sterilized 10 mM PBS. Containers were then placed on a shakertable for about two hours at 200 rpm. After shaking, the phosphatebuffered saline was then removed from each container.

E. Finally, to each container was then added approximately 1 L of 0.22mm filter sterilized water. Containers were then placed on a shakertable for about one hour at 200 rpm. After shaking, the water was thenremoved from each container.

Treated samples were cut and fixed for histological analyses.Hemotoxylin and eosin (H&E) and Masson trichrome staining was performedon both cross-section and long-section samples of both control andtreated tissues. Treated tissue samples appeared free of cells andcellular debris while control samples appeared normally and expectedlyvery cellular.

Example 2 Chemical Cleaning of Porcine Heart Valve

A porcine heart was procured from a 1 pound piglet and shipped inphysiological pH saline on ice. Within 4 hours, the heart valves wereremoved from the heart mass using scalpel and forceps. Some furthergross dissection was performed to remove excess tissue from around thevalves. One valve was retained as a control with sample pieces cut andfixed for various histological analyses while the other valve underwentthe chemical cleaning process. The remainder of the procedure wasperformed under aseptic conditions and at room temperature. The chemicalsolutions were all used at room temperature.

The valve was placed into 1 L solution of 100 mM EDTA/10 mM NaOH forabout 18 hours while agitating on a shaker platform. The valve was thenplaced into 1 L of 1 M HCl/1 M NaCl and agitated for 8 hours. The valvewas then placed into 1 L solution of 1 M HCl/10 mM phosphate bufferedsaline (PBS) and agitated for about 18 hours. The valve was then rinsedin PBS for between about 24 hours and then finally rinsed in sterilewater for about 1 hour while agitating. Treated sample pieces were thencut and fixed for various histological analyses.

Hemotoxylin and eosin (H&E) and Masson trichrome staining was performedon both cross-section and long-section samples of both control andtreated valves. Treated valve samples appeared free of cells andcellular debris while control samples appeared normally and expectedlyvery cellular.

Example 3 Chemical Cleaning of Porcine Artery, Pericardium and Fascia

A segment of femoral artery, the entire pericardium, and fascia wereprocured from a 450 lb. sow. The tissues were shipped in physiologicalpH saline on ice. The tissues were dissected further to remove excesstissue. Samples of each tissue were taken without cleaning for controlsamples and fixed for various histological analyses while the remainderof the tissues underwent the chemical cleaning process. The remainder ofthe procedure was performed under aseptic conditions and at roomtemperature. The chemical solutions were all used at room temperature.

The tissues were separately placed into 1 L solution of 100 mM EDTA/10mM and agitated on a shaker platform for about 18 hours. The tissueswere then each separately placed into 1 L solution of 1 M HCl/1 M NaCland agitated for 8 hours. Next, the tissues were separately placed intoa 1 L solution of 1 M HCl/1 mM phosphate buffered saline (PBS) and thenagitated for about 18 hours. The tissues were then separately rinsed inPBS for between about 2 to 4 hours and then finally rinsed in sterilewater for about 1 hour while agitating. Treated sample pieces were thencut and fixed for various histological analyses.

Hemotoxylin and eosin (H&E) and Masson trichrome staining was performedon both cross-section and long-section samples of both control andtreated tissues. Treated tissue samples appeared free of cells andcellular debris while control samples appeared normally and expectedlyvery cellular.

Example 4 Differently Ordered Chemical Cleaning

This procedure was performed under aseptic conditions and at roomtemperature and all chemical solutions were used at room temperature.

Mechanically stripped porcine intestine was cut into five 15 cm sectionsas described in example 1.

To each container was then added approximately 1 L of 0.22 mm filtersterilized solution of 1 M hydrochloric acid (HCl)/1 M sodium chloride(NaCl). Containers were then placed on a shaker table for between about6 to 8 hours at about 200 rpm. After shaking, the HCl/NaCl solution wasremoved from each container.

To each container containing intestine was added approximately 1 L of0.22 mm (micron) filter sterilized solution of 100 mMethylenediaminetetraacetic (EDTA)/10 mM sodium hydroxide (NaOH)solution. Containers were then placed on a shaker table for about 18hours at about 200 rpm. After shaking, the EDTA/NaOH solution wasremoved from each bottle.

To each container was then added approximately 1 L of 0.22 mm filtersterilized solution of 1 M sodium chloride (NaCl/10 mM phosphatebuffered saline (PBS). Containers were then placed on a shaker table forapproximately 18 hours at 200 rpm. After shaking, NaCl/PBS solution wasremoved from each container.

To each container was then added approximately 1 L of 0.22 mm filtersterilized solution of 10 mM PBS. Containers were then placed on ashaker table for about one hour at 200 rpm. After shaking, the phosphatebuffered saline was then removed from each container.

Finally, to each container was then added approximately 1 L of 0.22 mmfilter sterilized water. Containers were then placed on a shaker tablefor about one hour at 200 rpm. After shaking, the water was then removedfrom each container.

Treated sample pieces were then cut and fixed for various histologicalanalyses. Hemotoxylin and eosin (H&E) and Masson trichrome staining wasperformed on both cross-section and long-section samples of both controland treated tissues. Treated tissue samples appeared free of cells andcellular debris while control samples appeared normally and expectedlyvery cellular.

Example 5 Various Alkaline and Chelating Agents

The cleaning of mechanically stripped porcine intestinal submucosa wasfollowed as according to example 1. This procedure was performed underaseptic conditions and at room temperature and all chemical solutionswere used at room temperature. The chemical cleaning process of example1 was followed but the alkaline chelating agent of step A wassubstituted with other alkaline chelating agents of similar nature:

A. To each container containing intestine was added approximately 1 L of0.22 mm (micron) filter sterilized solution of either 100 mMethylenebis(oxyethylenitrilo)tetraacetic acid (EGTA)/10 mM NaOH; 100 mMEDTA/10 mM Ca(OH)2 (calcium hydroxide); or, 100 mM EDTA/10 mM K2CO3(potassium carbonate) solution. Containers were then placed on a shakertable for about 18 hours at about 200 rpm. After shaking, the alkalinechelating agents solution was removed from each bottle.

B. To each container was then added approximately 1 L of 0.22 mm filtersterilized solution of 1 M hydrochloric acid (HCl)/1 M sodium chloride(NaCl) solution. Containers were then placed on a shaker table forbetween about 6 to 8 hours at about 200 rpm. After shaking, the HCl/NaClsolution was removed from each container.

C. To each container was then added approximately 1 L of 0.22 mm filtersterilized solution of 1 M sodium chloride (NaCl)/10 mM phosphatebuffered saline (PBS). Containers were then placed on a shaker table forapproximately 18 hours at 200 rpm. After shaking, NaCl/PBS solution wasremoved from each container.

D. To each container was then added approximately 1 L of 0.22 mm filtersterilized solution of 10 mM PBS. Containers were then placed on ashaker table for about one hour at 200 rpm. After shaking, the phosphatebuffered saline was then removed from each container.

E. Finally, to each container was then added approximately 1 L of 0.22mm filter sterilized water. Containers were then placed on a shakertable for about one hour at 200 rpm. After shaking, the water was thenremoved from each container. Samples were fixed for histologicalanalyses.

Hemotoxylin and eosin (H&E) and Masson trichrome staining was performedon both cross-section and long-section samples of both control andtreated tissues. Treated tissue samples appeared free of cells andcellular debris while control samples appeared normally and expectedlyvery cellular.

Example 6 Various Acid and Salt Agents

The mechanically stripped porcine intestinal submucosa of example 1 waschemically cleaned using a substituted acid agent or substituted saltagent in step B. This procedure was performed under aseptic conditionsand at room temperature and all chemical solutions were used at roomtemperature.

A. To each container containing intestine was added approximately 1 Lsolution of 0.22 mm (micron) filter sterilized 100 mMethylenediaminetetraacetic tetrasodium salt (EDTA)/10 mM sodiumhydroxide (NaOH) solution. Containers were then placed on a shaker tablefor about 18 hours at about 200 rpm. After shaking, the EDTA/NaOHsolution was removed from each bottle.

B. To each container was then added approximately 1 L of 0.22 mm filtersterilized solution of either 1 M CH3COOH (acetic acid)/1 M NaCl or 1 MH2SO4 (sulfuric acid)/1 M NaCl solution. Containers were then placed ona shaker table for between about 6 to 8 hours at about 200 rpm. Aftershaking, the solution was removed from each container.

C. To each container was then added approximately 1 L of 0.22 mm filtersterilized 1 M sodium chloride (NaCl)10 mM phosphate buffered saline(PBS). Containers were then placed on a shaker table for approximately18 hours at 200 rpm. After shaking, NaCl/PBS solution was removed fromeach container.

D. To each container was then added approximately 1 L of 0.22 mm filtersterilized 10 mM PBS. Containers were then placed on a shaker table forabout one hour at 200 rpm. After shaking, the phosphate buffered salinewas then removed from each container.

E. Finally, to each container was added approximately 1 L of 0.22 mmfilter sterilized water. Containers were then placed on a shaker tablefor about one hour at 200 rpm. After shaking, the water was then removedfrom each container.

Treated sample pieces were then cut and fixed for various histologicalanalyses. Hemotoxylin and eosin (H&E) and Masson trichrome staining wasperformed on both cross-section and long-section samples of both controland treated tissues. Treated tissue samples appeared free of cells andcellular debris while control samples appeared normally and expectedlyvery cellular.

Example 7 Glycosaminoglycan (GAG) Content of ICL Determined by CelluloseAcetate Gel Electrophoresis and Alcian Blue Assay

To determine GAG content of ICL, cellulose acetate gel electrophoresiswith subsequent alcian blue stain was performed on extracts ofchemically cleaned ICL.

Samples of ICL underwent the chemical cleaning regimen outlined inExample 1, cut into 0.125 cm² pieces and placed into eppendorf tubes. Todigest the samples, 100 μl of papain (0.1 mg/ml papain in 0.1 M sodiumphosphate, 0.1 M sodium chloride, 0.005 M EDTA, 0.9 mg/ml cysteine, pH5.8) was added to each tube and allowed to incubate for about 18 hoursat 60° C. Standard containing known amounts of GAG (heparin) wereprepared in parallel. Dowex (0.4 g HCl form) and 3 ml water were thenadded. After spinning to remove the Dowex resin, 1 ml was removed andlyophilized. The samples were then rehydrated in 100 μl purified waterand centrifuged for about 5 minutes.

Samples were separated on cellulose-acetate sheets using the method ofNewton, et al. (1974). Cellulose-acetate sheets were soaked in 0.1 Mlithium chloride/EDTA buffer (pH 5.8) and blotted gently. Samples (5 μleach) were applied to the sheets at the cathode end and electrophoresedfor 30 minutes at 5 mA.

Following electrophoresis, the sheets were immersed immediately in analcian blue stain solution (0.2% alcian blue 8GX, 0.05 M magnesiumchloride, 0.025 M sodium acetate buffer (pH 5.8) in 50% ethylenealcohol) and placed on a shaker platform for about 30 minutes at roomtemperature. The sheets were then destained in at least three washes ofdestaining solution (0.05 M magnesium chloride, 0.025 M sodium acetatebuffer (pH 5.8) in 50% ethylene alcohol) for a total of about 30 minuteson a shaker platform. No detectable GAG staining was observed for papaindigested ICL while as little as 0.005 microgram heparin standard wasdetectable.

These results showed that the total amount of GAG remaining inchemically cleaned ICL is less than 1% (dry weight).

Example 8 Lipid Content of ICL Determined by Methylene ChlorideExtraction

ICL was laid out flat on plastic plates and air dried for two hours.Once dried, ICL was cut into smaller pieces of about 1 cm² of which1.100 g were transferred to a soxhlet thimble.

To a Kontes brand round bottom flask 24/40 was added 90 ml methylenechloride. The soxhlet was assembled in the fume hood with the bottom ofthe flask in a heated water bath and ice cooled water running throughthe distiller.

Extraction was allowed to proceed for four hours after which the soxhletwas disassembled. The round bottom flask containing the solvent andextracted material was left in the heated water bath until methylenechloride was evaporated until there remained 5 ml. The methylenechloride was then transferred to a 11×13 glass culture tube and theremaining solvent was boiled off. To the tube was added 2 ml ofmethylene chloride and the tube was capped immediately and the tubeplaced in a −20° C. freezer.

The weight of the extracted material was then determined. The glass tubewas placed in an ice bath. The weight of a Ludiag 1.12 ml aluminum weighboat was tared on a microbalance (Spectrum Supermicro). 10 μl ofresuspended extraction was added to the weigh boat and the solvent wasboiled off by placing the weigh boat on a hot plate for 45 seconds. Theweigh boat was allowed to cool for about 190 seconds and was placed onthe microbalance. The procedure was then repeated for extract volumes of20 μl and 30 μl.

Results indicate that the percentage of lipid is less than about 0.7%lipid by weight in dry chemically cleaned ICL. In contrast, nonchemicaUycleaned ICL contains a higher fraction of lipid; at least about 1.5% byweight in dry ICL that has not been chemically cleaned by the method ofthe invention.

Example 9 Amino Acid Analysis of ICL

Collagens are proteins characterized by their triple-helical regionswhich have a repeating triplet of amino acids glycine-X-Y, where X isfrequently proline and Y is often hydroxyproline. Hydroxyproline isfrequently used as an amino acid to identify and quantify collagens.Udenfriend, Science, 152:1335-1340 (1966).

To determine complete amino acid analysis of ICL, PICO-TAG HPLC wasperformed on mechanically cleaned (not chemically cleaned) porcine ICLand chemically cleaned ICL. Hydroxyproline content was measured for bothmaterials and compared.

Sample pieces of ICL from each condition weighing about from 0.31 toabout 0.36 g were dried further using a CEM AVC80 oven (CEM Corp.;Matthews, N.C.). Smaller samples were cut from these dried ICL piecesweighing about 9.5 to about 13.1 mg. Samples were placed into screw capculture tubes and the samples were then hydrolyzed (n=3 for eachcondition) in 1% phenol in 6 M HCl at 110° C. for about 16 hours. ICLhydrolysates were then diluted in 0.1 M HCl to normalize the materialconcentrations to 1 mg/ml. To labeled glass tubes (6×55), 20 ml ofhydrolysates and 8 ml of 1.25 mmol/ml L-norleucine as an internalstandard. Samples were then frozen and lyophilized. Samples were thenre-dried by adding 20 ml of 2:2:1 ethanol:water:triethylamine to thetubes, freezing and lyophilizing. Samples were then derivatized for 20minutes at room temperature by adding 20 ml of reagent (7:1:1:1ethanol:water:triethylamine:PITC) followed by freezing and lyophilizing.Samples were finally suspended in 200 ml PICO-TAG Sample Diluent andaliquoted to HPLC vials.

Amino acid standards were prepared in the following manner: 0.1 ml ofamino acid standard (Product #: A-9531, Sigma) was added to 1.9 ml 0.1 MHCl. Five serial dilutions at 1:1 were made using 0.1 M HCl. Volumes of100 ml for each serial dilution and 8 ml of 1.25 mmol/ml L-norleucinewere together added to glass tubes (6×55) and then prepared in the samemanner as ICL samples.

Samples and standards were run on a 3.9×150 mm PICO-TAG Amino Acidcolumn (Part# 88131; Waters Corp.; Milford, Mass.). Injections of 10 mlfor samples and 20 ml for standards were analyzed in triplicate foreach.

Results indicate for chemically cleaned ICL material, the content ofmajor collagenous amino acids in the material approach that of purifiedcollagen preparations. Using the hydroxyproline as a measure of collagencontent, the percentage of collagen by weight in ICL is calculated to beat least about 93% collagen by weight. In contrast, non-chemicallycleaned ICL contains a high fraction of non-collagenous amino acids;between about 11 to 25% by weight of ICL is non-collagenous material.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious to one of skill in the art thatcertain changes and modifications may be practiced within the scope ofthe appended claims.

We claim:
 1. A detergent-free and enzyme-free method for the removal ofnon-collagenous components from native mammalian tissue to yield anessentially collagenous tissue matrix, comprising: (a) contacting thetissue with a basic solution of ethylenediaminetetraacetic acid at a pHbetween about 8 and about 12; (b) contacting the tissue with an acidicsolution of sodium chloride at a pH between about 0 and about 1; (c)contacting the tissue with a solution of sodium chloride at a pH betweenabout 5 and about 9; and, (d) rinsing the tissue.
 2. A collagenoustissue matrix obtained by the method of claim 1.